Genomic context as well as sequence of both psr and penicillin-binding protein 5 contributes to β-lactam resistance in Enterococcus faecium

ABSTRACT Penicillin-binding protein 5 (PBP5) of Enterococcus faecium (Efm) is vital for ampicillin resistance (AMP-R). We previously designated three forms of PBP5, namely, PBP5-S in Efm clade B strains [ampicillin susceptible (AMP-S)], PBP5-S/R (AMP-S or R), and PBP5-R (AMP-R) in clade A strains. Here, pbp5 deletion resulted in a marked reduction in AMP minimum inhibitory concentrations (MICs) to 0.01–0.09 µg/mL for clade B and 0.12–0.19 µg/mL for clade A strains; in situ complementation restored parental AMP MICs. Using D344SRF (lacking ftsW/psr/pbp5), constructs with ftsWA/psrA (from a clade A1 strain) cloned upstream of pbp5-S and pbp5-S/R alleles resulted in modest increases in MICs to 3–8 µg/mL, while high MICs (>64 µg/mL) were seen using pbp5 from A1 strains. Next, using ftsW ± psr from clade B and clade A/B and B/A hybrid constructs, the presence of psrB, even alone or in trans, resulted in much lower AMP MICs (3–8 µg/mL) than when psrA was present (MICs >64 µg/mL). qRT PCR showed relatively greater pbp5 expression (P = 0.007) with pbp5 cloned downstream of clade A1 ftsW/psr (MIC >128 µg/mL) vs when cloned downstream of clade B ftsW/psr (MIC 4–16 µg/mL), consistent with results in western blots. In conclusion, we report the effect of clade A vs B psr on AMP MICs as well as the impact of pbp5 alleles from different clades. While previously, Psr was not thought to contribute to AMP MICs in Efm, our results showed that the presence of psrB resulted in a major decrease in Efm AMP MICs. IMPORTANCE The findings of this study shed light on ampicillin resistance in Enterococcus faecium clade A strains. They underscore the significance of alterations in the amino acid sequence of penicillin-binding protein 5 (PBP5) and the pivotal role of the psr region in PBP5 expression and ampicillin resistance. Notably, the presence of a full-length psrB leads to reduced PBP5 expression and lower minimum inhibitory concentrations (MICs) of ampicillin compared to the presence of a shorter psrA, regardless of the pbp5 allele involved. Additionally, clade B E. faecium strains exhibit lower AMP MICs when both psr alleles from clades A and B are present, although it is important to consider other distinctions between clade A and B strains that may contribute to this effect. It is intriguing to note that the divergence between clade A and clade B E. faecium and the subsequent evolution of heightened AMP MICs in hospital-associated strains appear to coincide with changes in Pbp5 and psr. These changes in psr may have resulted in an inactive Psr, facilitating increased PBP5 expression and greater ampicillin resistance. These results raise the possibility that a mimicker of PsrB, if one could be designed, might be able to lower MICs of ampicillin-resistant E. faecium, thus potentially resorting ampicillin to our therapeutic armamentarium for this species.

E nterococci are the second most common organism found in healthcare-associated (HA) infections, accounting for ~10%-15% of bacteria isolated from patients with these infections (1)(2)(3).Approximately, 80%-90% of clinical Enterococcus faecium (Efm) isolates are resistant to ampicillin (AMP-R) (4).When Efm strains are both AMP-R and vancomycin resistant, the two traditional drugs of choice, those infections can be very difficult to treat.Resistance to newer agents, such as linezolid and daptomycin, is also well documented and increasingly reported (5)(6)(7).
We now know that most clinical Efm isolates belong to a distinct Efm clade (clade A) that is evolutionarily separated by thousands of years from the community-associated (CA) clade B, comprising mostly human commensal isolates (8)(9)(10).Phenotypically, CA clade B is typically ampicillin susceptible (AMP-S) (MICs ranging from 0.125 to 2 µg/mL), while HA isolates are usually AMP-R (MICs often >64 µg/mL) (8,11).Penicillin-binding protein 5 (PBP5) from AMP-R strains of Efm is a low-affinity penicillin-binding protein that has been shown to be essential for AMP-R (12,13).There are two major and distinct PBP5 forms: PBP5-S, which is found in AMP-S CA clade B Efm, and PBP5-R, which is found in AMP-R HA clade A Efm (8,11).We have previously classified another form, PBP5-S/R, which appears to be a transitional form of PBP5 that is found in what has been designated (sub)clade A2 (closely related to clade A1 and commonly found in animals) (14).The pbp5-R and pbp5-S alleles differ by ~5% in their nucleotide sequence, and we previously reported a consensus of 20-21 amino acids (aa) (some PBP5-Rs have an additional aa) that can distinguish -S from -R proteins (11,14).A combination of four aa changes, made in PBP5-S/R from an AMP-S, (sub)clade A2 strain, was able to decrease the affinity of the resulting recombinant PBP5 protein for penicillin by 16-fold and increased the AMP MIC 5-fold (15).The generally accepted notion is that the intense pressure of β-lactam use in hospitals has selected for progressively more resistant strains via mutations in pbp5-S/R, resulting in more of the "R-specific" residues.Although we observed that hybrid PBP5-S/R patterns show a progression of amino acid changes from the S form to the R form of this protein, we could not strictly correlate these changes with ampicillin MICs (11,14).
Not only is the amino acid sequence of PBP5 important, but it appears that greater expression of Efm PBP5 is also important in mediating ampicillin resistance (16), yet it is still poorly understood.An early study of an AMP-R Efm strain (C68) found a homolog of psr from E. hirae (originally named as "PBP synthesis repressor") directly upstream of pbp5 (12).In E. hirae, the psr gene upstream of its pbp5 gene was concluded to contribute to penicillin resistance levels since an 87-bp deletion in the 5′ region of the psr ORF was associated with increased PBP5 levels and penicillin resistance: this led to the designation of this ORF as a repressor of pbp5 expression (17).In Efm, however, a role for Psr in pbp5 expression is not established.Rice et al. reported that Psr of the AMP-R HA strain C68 had an early stop codon, concluding that Psr was inactive and proposed that it did not participate in the regulation of pbp5 transcription or ampicillin resistance (12).
In our studies, to help delineate the role of Efm Psr, we previously reported that psr in HA clade A AMP-R Efm (psr A ) is truncated relative to psr in commensal AMP-S Efm (psr B ) (16).Specifically, high-ampicillin MICs were seen in strains with a 201-bp deletion in psr, consistent with the report mentioned above with E. hirae.This deletion encompasses 137 bp of the 3′ end of psr and 64 bp of the intergenic region of psr and pbp5.There are variations in deletions, and in some cases, there are insertions in the psr region in strains belonging to A1 or A2 cladal groups showing slight variations in high AMP MICs.Strain C68, as mentioned above (12), also has an insertion of one nucleotide in the psr-coding sequence resulting in an early stop codon (12), in addition to the missing downstream 201 bp.Thus, the presence of the early stop codon would likely not have any additional effect vs the effect of truncation.
Herein, we sought (i) to further explore the contributions of pbp5 sequence and clade background to AMP-R and (ii) to address our hypothesis that the presence of the longer ("full length") Psr seen in commensal AMP-S Efm (i.e., psr B ) leads to lower AMP MICs and repression of PBP5 expression relative to the presence of truncated psr typically found in HA strains.To test this hypothesis, we performed functional studies by cloning different pbp5 alleles downstream of various native or hybrid psr or ftsW/psr regions and ascertained the effects on AMP MICs and pbp5 expression.

Bacterial strains, plasmids, routine growth conditions, and susceptibility testing
Relevant characteristics of all Efm and Escherichia coli strains, newly created plasmid vectors, and pbp5, psr, and ftsW constructs used in the current study are described in Table S1; clinical Efm isolates and their AMP MICs are also described in Tables 1 to  4, while Fig. S1 shows a schematic representation of the pbp5 and the surrounding genes of clade B E. faecium strains COM15 and subclade A1 C68 (16).Efm D344SRF (18) was used as the host strain for all the plasmid constructs used in this study and is a derivative of a healthcare-associated (clade A1, original AMP MIC 24 µg/mL) clinical Efm that became AMP susceptible (MIC 0.023 µg/mL) following a 160-kb spontaneous genomic deletion that included pbp5, ftsW, and psr (18).pCWR plasmids were kindly provided by LB Rice and are described below and in footnotes of Tables 2 to 4 and Table S1.Naturally occurring pbp5-S, pbp5-S/R "hybrid, " and pbp5-R alleles encoding PBP5-S, PBP5 S/R, and PBP5-R and their previously published consensus amino acids residue profiles are described when used in the tables as well as Table S1; in this nomenclature, S indicates an amino acid that is part of the consensus sequence of 20-21 aa characteristic of AMP-S clade B strains, and R indicates an amino acid that is part of the consensus sequence characteristic of AMP-R clade A strains (11,14,19).Among these, the pbp5 alleles studied here came from two clade B Efm strains COM15 (S21/R0) (10,14,20) and E980 (S16/R5) (14,20,21) (referred to as pbp5 S and PBP5-S); two clade A2 Efm strains D366 (S7/R14) (11,14) and E1679 (S3/R18) (21) (pbp5 S/R encoding PBP5-S/R); and three clade A1 Efm strains C68 (S1/R20) (16,22), 1.231.502(S1/R20) (10,16), and TX82 (S0/R21) (16,23) Efm strains were grown at 37°C in brain heart infusion (BHI) (Becton, Dickinson (BD), Franklin Lakes, NJ) broth or agar with or without antibiotics as needed.E. coli strains were grown at 37°C using Luria-Bertani (LB; BD) broth or agar with or without antibiotics.AMP susceptibility testing was performed by broth microdilution in Mueller Hinton II (MH II) broth (cation adjusted; BD) following the Clinical and Laboratory Standards Institute guidelines (25) or by E-test (bioMérieux, France) for WT E. faecium strains.In the case of D344SRF and its derivatives with and without pbp5 plasmid constructs, MICs were performed on BHI agar via agar dilution method or with E-test since this strain grows very poorly on MH agar II.Kanamycin (KAN) and AMP were purchased from Sigma-Aldrich, St. Louis, MO.

Generation of pbp5 deletion mutants and pbp5 reconstituted in situ in the chromosome
Previously published methods by our group (26-29) were followed to construct chromosomal gene deletion mutants and to generate their chromosomal reconstitu ted/complemented constructs.To construct chromosomal pbp5-S deletion mutants, we used AMP-S clade B Efm strains: COM15 (16), which produces very little PBP5 and TX1330, which produces slightly more PBP5 than COM15 (16).Genomic DNA regions flanking the  pbp5 gene from 982 nucleotides (nt) upstream (UP fragment) and 1,142 nt downstream (DW fragment) were amplified by overlap extension PCR using primers containing NotI and BamHI restriction sites (Table S1) and cloned into NotI and BamHI sites of plasmid pHOU1 (16).pHOU1 contains a pheS* allele, encoding a phenylalanine tRNA synthetase with altered substrate specificity, which confers susceptibility to p-chloro-phenylalanine (p-Cl-Phe) and has a gentamicin (GEN) resistance marker (26)(27)(28)(29).The recombinant plasmid was then electroporated into the conjugative donor strain, E. faecalis CK111 (30), where it can replicate.The resulting donor strain was used in a conjugation experiment by filter mating with Efm strains COM15 and TX1330.Single-crossover integrants were selected on BHI plates containing gentamicin and erythromycin and then replated onto MM9YEG medium (M9-based medium supplemented with yeast extract, salts, and glucose) containing p-Cl-Phe (7 mM) to select for excision of pHOU1.We confirmed the excision of pHOU1 by the absence of growth on BHI + GEN 100-125 µg/mL agar plates, and colonies lacking pbp5 were detected by PCR.The correct pbp5 deletion was also confirmed by sequencing of the PCR fragment obtained by using outside primers specific for the flanking regions of the cloned gene and by pulsed-field gel electrophoresis to confirm the host identity following the methods we have previously described (26)(27)(28)(29).The resulting pbp5 deletion mutants COM15Δpbp5 and TX1330Δpbp5 were designated as TX6153 and TX6260, respectively (Table S1), and their AMP MICs were determined.
To restore the wild-type gene of TX1330Δpbp5 back into its native location in the chromosome, in brief, the pbp5 gene of TX1330 along with the upstream and down stream sequences was amplified using overlapping primers (Table S2), which ensured the removal of the BamHI site present at both ends of pbp5 gene, and then the above published methods were followed (26)(27)(28)(29) to create TX6262 (TX1330:Δpbp5::pbp5 TX1330 ).The pbp5 coding and promoter regions were sequenced to ensure that no unintended mutations had occurred, followed by determining the AMP MICs.We were unable to create a chromosomally complemented strain for COM15Δpbp5 as will be explained in the Results section.
For the deletion of pbp5-R from a clade A1 strain, we used Efm strain E1162 (S1/R20) and its previously published pbp5 deletion mutant E1162Δpbp5, which is a deletion of pbp5 after the first ~500 bp of the pbp5 gene (24).To complement E1162:Δpbp5, we followed the methods above using the pHOU1 system (26-29) and a PCR-generated fragment encompassing 66 bp upstream of the pbp5 start codon (including promoter region) plus 2,037 bp of the pbp5 E1162 -coding region plus 629 bp downstream of pbp5 sequence.The resulting strain, E1162:Δpbp5::pbp5 E1162 (TX6261), was tested for AMP MICs.

Construction and use of plasmid vectors for cloning various naturally occurring pbp5 alleles
To study the phenotypes of naturally occurring pbp5 alleles in D344SRF, we chose pCWR620 (kindly provided by Lou Rice) (15) which shows pbp5 expression levels similar to WT pbp5 for Efm strain C68 (16, 22) (Table S1).pCWR620 was originally designed to facilitate expression of pbp5-R from an AMP-R clade A1 Efm C68 cloned into an AMP-susceptible Efm strain D344SRF, which, as mentioned, lacks pbp5 (15).pCWR620 [also designated here pCWR620 (ftsW C68 /psr C68 )] contains ftsW and psr from Efm strain C68, from upstream of the ftsW promoter to downstream of the pbp5 promoter, inserted into the shuttle vector pTCV-lac (15,31).As we previously reported, C68 psr is truncated in pCWR620, relative to psr from clade B strains (16).In addition, it has a mutation in the coding region resulting in an early stop codon and is thought to be non-functional (15).To study various pbp5 alleles (S, S/R, and R) in this vector, we first PCR amplified pbp5 from different Efm strains, namely, two clade B Efm COM15 (S21/R0) and E980 (S16/R5); two clade A2 Efm D366 (S7/R14) and E1679 (S3/R18); three clade A1 Efm strains C68 (S1/ R20), 1.231.502(S1/R20), and TX82 (S0/R21).Designed PCR primers had BamHI restriction sites inserted on both ends which were used to clone amplified pbp5 fragments into pCWR620 (ftsW C68 /psr C68 ), followed by the transformation into E. coli DH5′ with selection on LB agar plates supplemented with kanamycin 50 µg/mL.After confirming the insert size and the nucleotide sequence, the recombinant plasmid DNA was electroporated into AMP-susceptible D344SRF lacking pbp5 with selection on Todd Hewitt agar plates containing 0.25M sucrose plus KAN 1,500 µg/mL, and resulting constructs were saved and are listed in Table S1.
The construction of ftsW A1 /psr A1 and ftsW B /psr B plasmids in pTCV-lac involved the use of two of our Efm strains, i.e., C68 (subclade A1) and COM15 (clade B), respectively.The configuration of their pbp5 and surrounding genes is illustrated in Fig. S1, sourced from our previously published paper (16).
Based on the AMP MICs obtained with the various constructs above, we decided to create four additional vectors using the same strategy as described above for pTEX6162 (ftsW COM15 /psr COM15 ) with a goal to study and compare AMP MICs of various pbp5 alleles with clade A1 and clade B psr, with and without their corresponding ftsW as well as cross-pairing (hybrid) of ftsW and psr in newly created vectors.In order to achieve this, we first created two hybrid vectors where clade A1 ftsW C68 was joined with clade B psr COM15 and vice versa via crossover PCR followed by cloning into pTCV-lac to generate pTEX6163 (ftsW C68 /psr COM15 ) and pTEX6164 (ftsW COM15 /psr C68 ), respectively.Second, we amplified psr alone with its promoter region from clade A1 E. faecium C68 (psr C68 ) and clade B E. faecium COM15 (psr COM15 ) with BamHI restriction sites in both ends, followed by cloning at the BamHI site of pTCV-lac; the resulting plasmids were designated as pTEX6172 (psr C68 ) and pTEX6173 (psr COM15 ), respectively (Table S1).All of the above mentioned various pbp5 alleles were cloned into pTCV-lac, result ing in pTEX6163 (ftsW C68 /psr COM15 ), pTEX6164 (ftsW COM15 /psr C68 ), pTEX6172 (psr C68 ), and pTEX6173 (psr COM15 ), followed by transformation into E. coli DH5′, extraction of recombinant plasmids, and then electroporation back into D344SRF.Resulting constructs are listed in Table S1 and Fig. 1, and their AMP MICs were determined and compared.
Additionally, we also analyzed two of the previously published plasmids, pCWR624 and pCWR666, carrying site-directed mutagenized pbp5 of clade A1 origin (15) in some of our pbp5 deletion mutants generated here, namely COM15Δpbp5 and TX1330Δpbp5, both belonging to clade B, and E1162Δpbp5 belonging to clade A1 (24), to evaluate and compare their AMP MICs vs the WT parent Efm strains COM15, TX1330, and E1162, respectively.In pCWR624, amino acid residues at positions 485, 499, and 629 in the active site region of PBP5 are methionine, isoleucine, and glutamic acid (typical of PBP5-S); in pCWR666, amino acids typical of PBP5-R are present at these sites, namely alanine, threonine, and valine, respectively, in addition to having serine at 466 position which is absent in pCWR624 (Table S1).The AMP MICs of these constructs in D344SRF have been reported earlier as 38 and 185 µg/mL, respectively (15).

Quantitative real-time PCR
For qRT-PCR analysis, three biological replicates per each test strain were used, and for each biological replicate, three technical replicates were used.We selected three representative constructs as described here to determine pbp5 expression levels.pbp5 derived from Efm TX82 (S0/R21) and cloned into pCWR620 (ftsW C68 /psr C68 ), i.e., TX6255 [D344SRF (pCWR620::pbp5 S0/R21_TX82 )] and in pTEX6162 (ftsW COM15 /psr COM15 ), i.e., TX6227 [D344SRF (pTEX6162::pbp5 S0/R21_TX82 )] was used.Control AMP-susceptible Efm strain D344SRF carrying the empty pTCV-lac vector was also used for baseline and for statistical significance.Bacteria were regrown from an overnight culture at 37°C with gentle agitation until an 0.8 OD 600 , and then cells were harvested.RNA extraction was done following standard methods (32) followed by synthesis of complementary DNA (cDNA) and evaluation of gene expression with 10 ng cDNA using SYBR Green (32).The gyrB Efm housekeeping gene (Table S2) was used for normalization.The intragenic pbp5 gene prime pair (Table S2) was used to determine pbp5 level of expression in all three test bacteria, and the primers were designed from TX82 pbp5 DNA sequence.Differential gene expression was analyzed with an unpaired (two-tailed) t test, and P < 0.05 was considered significant.

Generation of anti-rPBP5-R antibodies
Polyclonal antibodies against rPBP5-R (C68) were separately generated using a previously described scheme and as described earlier by us (16,33,34).Approved protocol and guidelines by the Animal Welfare Committee of The University of Texas Health Science Center, Houston, TX were followed.

PBP5 detection by western blotting
Cells grown overnight in BHI broth were inoculated into fresh BHI broth at a starting optical density at 600 nm (OD600) of 0.05 and grown at 37°C with gentle shaking until they reached an OD 600 of ~0.1-0.2.The cultures were centrifuged at 3,900 rpm for 10 min, and the pellets were rapidly chilled on dry ice.Cells were washed with 0.02 M Tris-HCl, pH 7.0, and 0.01 M MgSO 4 buffer and resuspended in one-tenth the volume of the same buffer containing 100 µM PMSF.Following the addition of mutanolysin to a final concentration of 10 U/1 OD 600 of cells, tubes were incubated at 37°C for 1 h in a rotating shaker, followed by centrifugation to recover the supernatant (16,33).Protein concentrations were measured as described previously (16,33).Samples were normalized so that the total protein concentrations were equally followed by mixing (1:1) in 2× Laemmli sample buffer (BIO RAD, CA).Samples were treated at 100°C for 10 min prior to separating them by 4%-20% Mini-PROTEAN TGX Precast Protein Gels (BIO RAD, CA) under reducing conditions in running buffer and transferred to a nitrocellulose membrane following the manufacturer's protocol.Membranes were then probed with rPBP5-R (C68) polyclonal rat sera preabsorbed for 2 h with cell lysates of D344SRF in order to remove non-specific background bands, followed by HRP-conjugated goat anti-rat IgG antibodies (secondary antibody) and developed using Bio-Rad's chemilumi nescent detection reagents (BIO RAD, CA) per manufacturer's instructions.

AMP susceptibility testing of pbp5 deletion mutants and their derivatives with pbp5 reconstituted in situ in the chromosome
The construct details and AMP MICs are shown in Table 1.Deletion of pbp5 from the clade B E. faecium strain COM15 resulted in only a small decrease (two-to threefold) in AMP MIC (0.06 µg/mL for COM15Δpbp5) vs MIC (0.12-0.19 µg/mL for its parental wild type) (Table 1); the small decrease is consistent with our earlier observation that this strain produces very little if any PBP5 (16).On the other hand, another clade B strain TX1330, previously shown to produce more PBP5 than WT COM15 in western blots (16), showed a greater reduction in AMP MIC when its pbp5 was deleted (from AMP MIC 1-1.5 µg/mL for WT to 0.01-0.09µg/mL for Δpbp5) (Table 1).The in situ reconstituted strain, TX1330Δpbp5::pbp5 TX1330 , showed an AMP MIC similar to that with WT TX1330 (Table 1).The AMP MIC of the published clade A1 partial pbp5 deletion mutant E1162Δpbp5 was 0.25 µg/mL (24); when reconstituted in situ with pbp5 from the parental E1162, AMP MICs were 32 µg/mL, similar to the WT E1162 (Table 1).
The plasmid construct designs and MIC results are shown in Fig. 1 and Tables 2  to 5, respectively.To evaluate the effect on AMP MICs generated by pbp5 alleles from strains belonging to different clades and that show different AMP MICs and different proportions of "S" amino acids and "R" amino acids, we cloned pbp5 alleles into the previously published vector pCWR620 (ftsW C68 /psr C68 ); this vector (kindly provided by LB Rice and derived from pTCV lac) has ftsW and psr from C68, a (sub)clade A1, AMP-R strain (15).The AMP MICs of the host strain E. faecium D344SRF and the various plasmid constructs and WT strains are shown in Table 2.The AMP MICs of D344SRF harboring the empty vector pTCV-lac showed no change vs D344SRF alone (0.023 µg/mL) (Tables 2 and  3).E. faecium D344SRF derivatives harboring pCWR620 pbp5-R alleles from AMP-R, clade A1 strains were all highly AMP-R, with MICs similar to the respective WT strains that were the source of the pbp5 allele, namely C68 (AMP 128 µg/mL), 1.231.502(AMP 128-256 µg/ mL), and TX82 (AMP 64 µg/mL) and one clade A2 strain E1679 (AMP 256 µg/mL) (Table 2).
On the other hand, D344SRF harboring pCWR620 plasmid with pbp5 alleles from strains with lower AMP MICs (E980 and COM15) showed moderate AMP MICs ranging from 3 to 8 µg/mL.The differences between AMP MICs generated with pbp5-R alleles vs pbp5-S and pbp5-R/S alleles would be expected (15) and reflect the greater number of those amino acids previously associated with and/or experimentally shown to influence AMP MICs (14,15,18).While the AMP MICs generated by the constructs with pbp5-S and pbp5-R/S alleles were somewhat higher than the AMP MICs of the respective WT strains from which these alleles originated, this is likely due to plasmid copy number generating more copies of PBP5.

Influence of ftsW/psr from different clades on AMP MICs generated by various naturally occurring pbp5 alleles cloned in trans into E. faecium D344SRF
Since pCWR620 (15) used above contains ftsW/psr from a clade A1, AMP-R strain (C68), we next investigated the influence of ftsW/psr from a clade B, AMP-S strain on AMP MICs generated by the different pbp5 alleles (Table 3).To achieve this, we cloned ftsW/psr from COM15 (clade B; AMP MIC 0.12-0.19µg/mL) into pTCV-lac resulting in pTEX6162 and then inserted different pbp5-R alleles into this vector, as above.The resulting derivatives with four different pbp5-R alleles showed considerably lower AMP MICs (range 4-16 µg/mL) than their WT parent clade A1 strains [C68, 1.231.502,TX82, and clade A2 strain E1679 (AMP MIC generally >64 µg/mL)] (Table 3) and lower MICs than constructs with these pbp5 alleles cloned into the same vector backbone (pCWR620) downstream of the clade A ftsW C68 /psr C68 (AMP MIC range, 64-256 µg/mL).We also inserted pbp5-S alleles from three different AMP-S strains into pTEX6162 (ftsW COM15 / psr COM15 ).The resulting strains showed considerably lower MIC than when these alleles were downstream of the clade A ftsW C68 /psr C68 in pCWR620 (Table 3).Also, as above, while slightly increased AMPs (MIC range of 0.5-3 µg/mL) were seen vs their respective WT parent strains (Table 3), this is likely due to plasmid copy number.
To look further at the roles of psr and ftsW, we generated additional plasmid constructs derived from pTCV-lac with psr-only from each clade (without ftsW) and hybrid constructs with ftsW from one clade and psr from the other.AMP MICs for derivatives of D344SRF with these plasmid constructs and various pbp5 alleles (Table 4) showed that AMP MICs were low with clade A1 ftsW C68 plus clade B psr COM15 (third column, Table 4) as well as with clade B psr COM15 alone (second column, Table 4) and were approximately equal to those when both psr and ftsW were from clade B (last column, Table 3).On the other hand, with the clade B ftsW COM15 plus the clade A1 psr C68 hybrid construct (last column Table 4) as well as with clade A1 psr C68 alone (first column, Table 4), AMP MICs generated by the different pbp5 alleles were much higher and approximately equal to AMP MICs seen when both psr and ftsW were from clade A (middle column, Table 3).a Range of MICs reflects results with colonies and/or repetitions.b pCWR624 kindly provided by LB R 47ice is pCWR620 with pbp5 (S7/R14) from clade A2 strain D366 mutagenized to contain methionine, isoleucine, and glutamic acid at positions 485, 499, and 629 in the active site of PBP5.No serine at position 466 (15).c pCWR666 kindly provided by LB Rice is pCWR620 with pbp5 from clade A1 strain C68 (S1/R20) previously mutagenized to contain serine at 466 position in addition to having methionine, isoleucine, and glutamic acid at positions 485, 499, and 629 in the active site of PBP5 (15).
We next explored the effect on AMP MICs when plasmids pCWR624 and pCWR666 [both of which have clade A1 ftsW A1 /psr A1 from C68 with downstream pbp5 alleles previously engineered (15) to express high levels of AMP-R] were introduced into strains deleted for pbp5 but which retained their own psr and ftsW (Table 5).In contrast to the high AMP MICs seen when these plasmids were introduced into D344SRF (a clade A1 strain lacking pbp5, psr, and ftsW), AMP MICs were low (2-4 µg/mL) when introduced into each of the clade B strains (COM15Δpbp5 and TX1330Δpbp5).One possible explanation for this difference is that the presence of the endogenous clade B psr results in greater susceptibility to AMP regardless of the presence of psr A1 ; however, an effect of some other component of the clade B host background is certainly possible.In contrast, as expected, high MICs (128-256 µg/mL) resulted when these plasmids were introduced into the clade A1 strain E1162Δpbp5 (Table 5) with both a cis and a trans copy of psr A1 .

FtsW amino acid sequence alignments
The comparison of FtsW amino acid sequence alignments revealed an 8/387 amino acid difference between clade A1 and clade B strains.Within the subset of clade A1 strains examined, C68, TX82, and E1162 shared a 100% identical sequence, whereas TX16 exhibited a variance of 1/387 amino acids, and TX1.231.502 had an 11/387 amino acid deletion.Among clade B strains (COM15, E980, TX1330), there were only 3/387 amino acids that differed.These findings collectively indicate a generally high conservation of the FtsW amino acid sequence (Fig. S2).

PBP5 detection by western blotting
The impact of ftsW/psr from different clades on cloned pbp5 (from TX82, clade A) in trans into E. faecium D344SRF was assessed through western blotting.Analysis using a polyclonal serum raised against recombinant PBP5-R (rPBP5-R) from strain C68 revealed higher PBP5 protein levels in plasmids carrying the clade A psr C68 , irrespective of the ftsW origin or presence (Fig. 3).This observation again suggests that ftsW does not contribute to the observed increase in PBP5 levels on western blots.Likewise, TX82 pbp5 (S0/R21) in plasmids containing psr COM15 exhibited low PBP5 levels, regardless of the ftsW origin or presence, also supporting that ftsW appears not to influence the PBP5 levels or MICs detected in western blots (Fig. 3).The MICs of constructs without ftsW displayed MICs equal to or at most slightly lower than those with ftsW (32-64 µg/mL vs 128-256 µg/mL for clade A gene(s) and 4-6 µg/mL vs 8 µg/mL for clade B strains) (Tables 3 and 4; Fig. 3).These differences are within the error expected in MIC.
We also observed that in the case of pbp5 cloned downstream of ftsW C68 /psr C68 (lane 2, Fig. 3) with AMP MIC 64/128, the western blot analysis revealed a comparatively lower PBP5 presence than that observed with the same pbp5 cloned, in the same vector background, downstream of ftsW COM15 /psr C68 or psr C68 (lanes 4, 5, Fig. 3) despite these later constructs generating lower AMP MICs (24 and 32 µg/mL, respectively).No discernible differences in the levels of the RNA polymerase β subunit were noted except in lane 5 which might have occurred due to unknown/technical reason.
The lack of a strict correlation between protein levels seen with pbp5 with ftsW C68 / psr C68 (lane 2, Fig. 3) vs the same pbp5 with ftsW COM15 /psr C68 or psr C68 (lanes 4, 5, Fig. 3) is not particularly surprising, as posttranscriptional, translational, and posttranslational regulatory networks are known to influence protein abundance (16,35).Previous studies have demonstrated instances where genes with similar mRNA levels exhibited up to a 20-fold difference in protein abundance (36).In our prior research involving a separate published study, there was not complete agreement observed between PBP5 protein levels, pbp5 mRNA levels, and their corresponding AMP MICs (16,35).It is also conceiv able that hybrid ftsW/psr combinations may in some unknown way impact the AMP MICs, perhaps via an interaction with the PBP5 target proteins by ftsW C68 /psr C68 , which exhibited a higher AMP MIC range.

DISCUSSION
Historically, high-level AMP resistance in Efm has been attributed primarily to amino acid sequence changes in PBP5 (11,14,15,37).It has been demonstrated that specific pbp5 alleles are associated with specific Efm clades and that some specific substitutions found in clade A1 PBP5s result in a decrease in the affinity of this protein for penicillin and in higher AMP MICs (16).However, pbp5 sequence variation alone has not been able to explain the range of MICs seen in Efm (11,14), and expression differences have also been demonstrated (16).The region upstream of pbp5 has been suggested to contribute to levels of AMP resistance (17), although a direct role of the upstream Psr in pbp5 expression has been debated (12,15).Previously, we reported evidence of considerable variance in the ftsW/psr region immediately upstream of the pbp5 gene, including DNA fragment insertions and deletions in this region, among Efm strains from different clades and with different ranges of AMP MICs; these differences generally appeared to correlate with AMP MICs and the differential abundance of PBP5, with clade A strains consistently having a truncated or interrupted psr relative to psr from clade B strains (16).
The results presented in this study agree with prior observations that allele differences in pbp5 are important for AMP MICs.We show the effect of various naturally occurring pbp5 alleles from clades A1, A2, and B on the AMP MIC when cloned and introduced in trans into Efm strain D344SRF, lacking the region encompassing ftsW, psr, and pbp5.D344SRF derivatives harboring pbp5 alleles from AMP-R clade A1 strains cloned into a vector with upstream clade A1 fstW/psr were all highly AMP-R, with MICs similar to the respective WT strains (Table 2).On the other hand, D344SRF with the same vector containing cloned pbp5 alleles from AMP-S or intermediate clade B and A2 strains showed low to moderate levels of AMP MICs (Table 2).We also noted that plasmids with pbp5-S and -R/S alleles resulted in higher AMP MICs than seen with their respective WT strain.This could be due to plasmid copy number resulting in the production of more PBP5, the impact of the upstream clade A1 psr or ftsW in the vector, or other host factors.Zhang et al., for example, reported additional genes within Efm genomes that contribute to elevated ampicillin MICs (24).
Most importantly, our results point to the importance of psr in modulating AMP resistance in Efm.Specifically, the origin and genomic context/sequence of the ftsW/psr region were shown to be important for variations seen among AMP MICs (Tables 3 and  4).By cloning combinations of ftsW ± psr from different clades together with distinct pbp5-S, R/S, or R alleles into D344SRF (which lacks its own ftsW/psr/pbp5), we have shown that the presence of psr B leads to lower expression of PBP5 and lower MICs compared to the presence of psr A , regardless of which pbp5 allele is present.Specifically, in the presence of clade A1 psr [pCWR620 (ftsW C68 /psr C68 )] and different pbp5 alleles, the AMP MICs were considerably higher (Tables 2 and 3) than in the presence of clade B psr [pTEX6162 (ftsW COM15 /psr COM15 )].As shown in Table 4, having ftsW from a differing clade than psr had little effect on the AMP MICs.Moreover, psr B and psr A cloned alone with their own promoter, without ftsW, resulted in the same phenotype as psr cloned with an ftsW allele (Table 4).These results indicate that psr alone can account for the differences in AMP MIC seen with the ftsW/psr hybrid clones and that ftsW does not appear to play a considerable role under these conditions.Western blot analysis of PBP5 expression not only confirmed the influence of psr on PBP5 but also suggested that ftsW is not a significant contributing factor to the observed protein level variation (Fig. 3).
When considering the implications of these results, one consideration is that it might be possible to generate compounds to mimic a Psr B effect to decrease PBP5-R expression and lower Amp MICs as a possible avenue for new antimicrobials.Whether AMP MICs of WT clade A1 strains which have a WT-truncated psr A would be lowered by a Psr B -mimic is not clear, as we were unable to successfully introduce stable plasmids containing psr B into WT clade A1 strains that contain their endogenous ftsW/psr/pbp5.However, we were able to show (Table 5) that a clade A1-truncated psr A (present in the pCWR plasmids as ftsW C68 /psr C68 /pbp5 C68 ) does not lead to high MICs when placed in the clade B strains COM15Δpbp5 and TX1330Δpbp, which still have their chromosomal psr B , i.e., the presence of clade A psr did not overcome an effect of the presence of clade B psr.If other host background influences were to be ruled out, this would imply that Psr B is "dominant" to Psr A since the presence of both A and B psr alleles (Table 5) in derivatives of COM15Δpbp5 and TX1330Δpbp5 still resulted in low MICs.
The mechanism for the influence of Psr on pbp5 expression and AMP MICs is yet to be determined.Psr (renamed LcpA for E. hirae Psr) belongs to the LytR-CpsA-Psr family (38).This family of proteins is found in most gram-positive bacteria, and some contain more than one Lcp protein (39).This protein family is poorly understood, and a multitude of functions has been reported, including cell surface properties, virulence, antibiotic resistance, and septum formation (40)(41)(42)(43).Structural and domain analysis of Psr sequences revealed the presence of an HTH domain and a transmembrane domain, likely as domains associated with transcriptional attenuation.However, in the present study, we did not perform any experiments showing that Psr acts as repressor protein or regulates the PBP5 expression by binding to an operator DNA sequence.This remains a subject for our future studies.In E. hirae, evidence suggests that LcpA plays a role in cell wall metabolism, "probably acting as a phosphotransferase catalyzing the attachment of cell wall polymers to the peptidoglycan" (40).Thus, the mechanism by which Psr B lowers AMP MICs and PBP5 expression may be indirect, perhaps by a cell wall metabolism effect resulting in feedback repression of PBP5 expression, while truncated and presumably inactive Psr A does not.Such a phenomenon is not without precedent since it has been reported in other organisms that the nature of the peptidoglycan precursors can alter the level of β-lactam resistance associated with the expression of low-affinity PBPs (44)(45)(46).
Overall, the results presented in this study provide further insight into ampicillin resistance of E. faecium clade A strains, highlighting that, in addition to amino acid sequence alterations in PBP5, the psr region is important for the expression of PBP5 and AMP resistance.The presence of full-length psr B results in lower expression of PBP5 and lower MICs than does the presence of a shorter psr A , regardless of the pbp5 allele present (Table 4).Lower AMP MICs were also seen when psr alleles from each clade were present together in clade B strains, although an effect of other differences between clade A and B strains may also contribute.It is interesting that, in the split between clade A and clade B E. faecium and the further evolution of higher AMP MICs in hospital-associated strains, changes in pbp5 appear to have co-evolved with changes in psr that appear to have resulted in an inactive Psr, allowing higher levels of expression together with more ampicillin-resistant PBP5.Further studies are warranted in order to elucidate the underlying mechanism for the differential regulation of PBP5 and ampicillin resistance by psr between the Efm clades.

TABLE 4 a
Effect of psr alone and of hybrid ftsW/psr constructs from an AMP-S clade B strain and an AMP-R clade A strain on AMP MICs generated by pbp5 alleles cloned from different E. faecium clades in the same pbp5-lacking background a Strain origin of cloned pbp5 alleles (aa profile b and clade) Host strain D344SRF used for all plasmid constructs is a healthcare-associated (clade A1) E. faecium and has a 160-kb spontaneous genomic deletion encompassing pbp5, ftsW, psr regions (15).b Number of amino acids characteristic of PBP5-S consensus (S)/number of aa characteristics of PBP5-R consensus (R)(11,14).c Host plasmid pTEX6172 (this study) is derived from the shuttle vector pTCV-lac and contains psr A1 from clade A1 E. faecium strain C68 which has a deletion of 201 bp relative to clade B psr alleles and a premature stop codon.d Host plasmid pTEX6173 (this study) is derived from the shuttle vector pTCV-lac and contains psr B from E. faecium COM15 and does not have the 201-bp deletion or the premature stop codon found in psr C68 .e Host plasmid pTEX6163 (this study) is derived from the shuttle vector pTCV-lac and contains ftsW A1 from AMP-R, clade A1 E. faecium C68, and psr B was from AMP-S, clade B E. faecium COM15.f Host plasmid pTEX6164 (this study) is derived from the shuttle vector pTCV-lac and contains ftsW B from AMP-S, clade B E. faecium strain COM15, and psr A1 was from AMP-R, clade A1 E. faecium C68.g Range of AMP MICs reflects results with different colonies and/or repetitions.

FIG 1
FIG 1 Illustrative depiction of the plasmid vectors, derived from pTCV-lac, for investigating effects of different pbp5, ftsW, and psr alleles.The expression vector pTCV-lac was employed for cloning ftsW and psr alleles from clade A1 (C68) and clade B (COM15) E. faecium strains, as well as through cross-pairing (hybrid) of ftsW and psr from these clades, at the SmaI-BamHI site.The ftsW/psr segments are visually represented in blue (COM15) and red (C68).The host strain, D344SRF, is a healthcare-associated clade A1 E. faecium with a prior spontaneous genomic deletion spanning 160 kb, which includes the pbp5, ftsW, and psr regions.pCWR620 (15)-ftsW A1 /psr A1 from clade A1 strain C68 exhibits a naturally truncated psr and has a mutation in the coding region, leading to the presence of an early stop codon.pTEX6162-ftsW B / psr B is from COM15; it has full length psr.pTEX6163-ftsW A1 is from C68, and psr B (full length) is from COM15.pTEX6164-ftsW B is from COM15, and psr A1 (truncated with premature stop codon) is from C68. pTEX6172-psr A1 (truncated with premature stop codon) is from C68. pTEX6173-psr B (full length) is from COM15.

FIG 2
FIG 2Quantitative real-time PCR for pbp5 expression.Comparative expression levels (log 2 fold) of the pbp5 A1 gene from strain TX82 cloned into strain D344SRF using pTCV-lac (empty vector), pCWR620 which has ftsW A1 /psr A1 from clade A1 Efm strain C68 upstream of pbp5 TX82 , and pTEX6162 which has ftsW B /psr B from clade B strain COM15.Three biological replicates per test bacteria were used, and for each biological replicate, three technical replicates were used.

TABLE 1
Origin and AMP MICs of WT strains, pbp5 deletion mutants, and derivatives with the WT pbp5 reconstituted in its native chromosomal location

TABLE 2
AMP-MICs conferred by pbp5 alleles from different E. faecium clades cloned in trans into the same pbp5-lacking background e

Strain origin of cloned pbp5 alleles (PBP5 amino acid profile a and clade) AMP MICs (µg/mL) Wild-type E. faecium D344SRF c ± pCWR620 b,c containing the pbp5 allele from the WT strains in the first column
d NA, not applicable (see c ). e NA, not applicable since this strain has no pbp5 to clone.

TABLE 3
Effect of ftsW/psr from clade A and clade B on AMP MICs conferred by pbp5 alleles cloned from different E. faecium clades into the same pbp5-lacking background f

Strain origin of cloned pbp5 alleles (aa profile a and clade) [AMP MIC (µg/mL)] AMP MICs (µg/mL) pbp5 allele from first column cloned downstream of clade A1 ftsW C68 /psr C68 b pbp5 allele from first column cloned downstream of clade B ftsW COM15 /psr COM15 c
(15,16)is derived from the shuttle vector pTCV-lac and contains ftsW B and psr B from the AMP-S clade B E. faecium strain COM15; this psr does not have the 201-bp deletion or the premature stop codon seen in psr of C68(15,16).
c pTEX6162 (this e Range of MICs reflects results with different colonies and/or repetitions.f NA, not applicable since this strain has no pbp5.

TABLE 5
(12)uence of host background on AMP MICs generated by pbp5 alleles previously generated(12)to express higher levels of AMP resistance